CN106226515B - A kind of method of quick detection Listeria Monocytogenes - Google Patents

A kind of method of quick detection Listeria Monocytogenes Download PDF

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CN106226515B
CN106226515B CN201610693822.1A CN201610693822A CN106226515B CN 106226515 B CN106226515 B CN 106226515B CN 201610693822 A CN201610693822 A CN 201610693822A CN 106226515 B CN106226515 B CN 106226515B
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CN106226515A (en
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赵广英
窦文超
马俊爽
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Zhejiang Gongshang University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria

Abstract

The invention discloses a kind of methods of quick detection Listeria Monocytogenes, include mainly step：Prepare Fe3O4/SiO2–NH2Nanoparticle prepares Ab/SiO2/Fe3O4, prepare rGO/AuNPs/Ab, double-antibody method and prepare immunosensor, DPV detections and result and judge.The present invention using redox graphene be the fixed AuNPs of substrate as electrochemical label, using immune magnetic Nano microsphere as capture probe, for being enriched with the object in measuring samples.To modify AntiL.monocytogenesRedox graphene/nanogold of antibody is as non-enzyme marker and enrichmentL.monocytogenesIMNPS formed interlayer structure.Interlayer structure compound is adsorbed on screen printing carbon electrode surface under the action of externally-applied magnetic field, is analyzed with differential pulse voltammetry.It is y=0.02933x 0.01996, R that DPV response signals have good linear relationship, equation of linear regression in 109 CFU/mL of 103CFU/mL2=0.99367, detection is limited to 1.8 × 104 CFUmL‑1(S/N=3), i.e., 54 CFU in the detection sample of 3 μ L in the detectionL.monocytogenesIt can be detected.

Description

A kind of method of quick detection Listeria Monocytogenes
Technical field
The invention belongs to antibiotic in food to remain rapid detection technical field, and especially one kind is with reduction-oxidation graphite Alkene/nano-Au composite is used to quickly detect the side of Listeria Monocytogenes as the immunosensor that non-enzymatic marks Method.
Background technology
Listeria Monocytogenes (Listeria monocytogenes, L.monocytogenes) are used as one Kind mankind's food-borne pathogens, are prevalent in poultry, meat and dairy products.It can lead to gastroenteritis after the mankind are infected, lose Mass formed by blood stasis, meningitis, miscarriage are easier to occur to pregnant woman, baby, the elderly and immunocompromised person.Having in the infected causes 25%-30% lethalities.So particularly important to the quickly detection of L.monocytogenes in food.
Electrochemical immunosensor (Electrochemical Immunosensor) has well as a kind of in recent years The advantages such as selective, highly sensitive, simple and inexpensive are concerned, wherein non-enzymatic mark electrochemistry immunosensor (Non- Enzymatic Electrochemical Immunosensors) and traditional enzyme mark electrochemistry immunosensor (Enzymatic Electrochemical Immunosensors) is compared, the former can not only ensure high susceptibility and sound Rapid advantage is answered, while overcoming traditional enzyme mark electrochemistry immunosensor kind since enzyme is originally experienced temperature, pH value etc. It influences, in some special chemical environments, the easy loss of activity of enzyme and the disadvantage that keeps immunosensor unstable.So more next More Research tendencies improves sensitivity and steady in simulating the signal catalytic amplification of enzyme in enzyme labeled immunoassay sensor It is qualitative.(such as Pt-Pb) and graphene oxide material are combined with monometallic nano material (such as nanogold, nano silver), more metals It is marked as non-enzymatic and generates signal, reach testing goal.
Invention content
In order to overcome drawbacks described above in the prior art, the present invention provides a kind of quickly detection monocyte hyperplasia Li Si The method of special Salmonella.
A kind of method of quick detection Listeria Monocytogenes, which is characterized in that include the following steps：
(1) Fe is prepared3O4/SiO2–NH2Nanoparticle：FeCl3It is dissolved in distilled water, FeSO is added4, it is eventually adding NH3·6H2O, magnetic agitation 60min, obtains Fe under anaerobic3O4Magnetic Nano material；It is added in the reaction vessel anhydrous Ethyl alcohol, distilled water, TEOS and NH3·6H2O is stirring evenly and then adding into Fe3O4Magnetic Nano material is stirred for 24 hours at 25 DEG C, is obtained Fe3O4/SiO2Magnetic Nano material；Absolute ethyl alcohol, APTES and Fe are added in the reaction vessel3O4/SiO2Magnetic Nano material； It is stirred at 25 DEG C for 24 hours, obtains Fe3O4/SiO2–NH2Nanoparticle；
(2) Ab/SiO is prepared2/Fe3O4：Fe3O4/SiO2–NH22.5% glutaraldehyde is added in nanoparticle, and 25 DEG C are protected from light magnetic Power stirs 3h, is added Anti-L.Monocytogenes (Ab) after amino-reactive, is closed with BSA after stirring 2h at 25 DEG C non-specific Property binding site, obtains Ab/SiO2/Fe3O4(IMNPS)；
(3) rGO/AuNPs/Ab is prepared：Graphene oxide is dissolved in distilled water, and HAuCl4 is added, is heated to boiling, rapidly Sodium citrate reaction is added, obtains rGO/AuNP nano materials；Anti- is added into rGO/AuNPs nano materials L.monocytogenes (Ab), 12h is incubated at 4 DEG C, is closed nonspecific binding site with BSA, is obtained rGO/AuNPs/Ab and receive Rice material；
(4) double-antibody method prepares immunosensor：Ab/SiO obtained by step (2) is added in testing sample solution2/ Fe3O4(IMNPS), 40min is incubated at 25 DEG C after mixing, PBS is washed, and obtains IMNPS immuno absorbences L.monocytogenes Compound, add rGO/AuNPs/Ab obtained by step (3), be incubated 40min at 25 DEG C after mixing, PBS washings obtain rGO/AuNPs/Ab/L.monocytogenes/IMNPS；RGO/AuNPs/Ab/L.monocytogenes/IMNPS is taken to be added drop-wise to Screen printing electrode working electrode surface, while magnet is placed at the working electrode back side, ensure rGO/AuNPs/Ab/ L.monocytogenes/IMNPS compounds are firmly adsorbed on working electrode surface, prepare immunosensor；
(5) DPV is detected：100 μ L0.2M HCl are added dropwise in working electrode surface and test bottom liquid, carry out DPV detections, wherein work It is immunosensor electrode to make electrode, is carbon electrode to electrode, and reference electrode is Ag/AgCl electrodes, is characterized and is determined with DPV Property judges immunoelectrode；The setting of DPV detection methods：Oxidizing potential 1.25V, oxidization time 120s, DPV electric potential scanning are ranging from 1.0V-0.0V；Obtain peak point current y；
(6) result judges：Peak point current y is substituted into formula:Y=0.02933x-0.01996 is calculated L.Monocytogenes bacteria concentration x values.
Since nanogold (AuNPs) can be by simple chemical method Fast back-projection algorithm, uniform particle sizes, specific surface area is high, Good conductivity facilitates label biomolecule, is passed to make nanogold be widely used in electro-chemistry immunity as signal amplified material Sensor.Graphene has good thermal conductivity and high conductivity, simultaneously because the carbon nano-structured of its two dimensional surface can be used as admittedly The substrate of deposit metal nanometer material further makes metal nano material to make the amount of metal nano material in unit area increase The catalytic amplification of material increases.In the present invention, using sodium citrate as one step redox graphene of reducing agent and chlorine gold Acid obtains redox graphene/nanogold (rGO/AuNPs) nano material compound, using differential pulse voltammetry (Differential Pulse Voltammetry, DPV) AuNPs using in rGO/AuNPs is as signal amplified material, and principle is such as Under：AuNPs is oxidized to AuCl in the liquid of the bottoms HCl with constant voltage-4, AuCl under the conditions of DPV-4It is reduced into Au0, this process Response signal is produced, reduction peak to peak current is obtained.To further increase electric signal on the basis of rGO/AuNPs, spirit is improved Sensitivity, using immune magnetic Nano microsphere high-specific surface area, magnetic conductance tropism, easily concentration and separation object etc. is excellent from complex sample Point, for building electrochemical immunosensor.The present invention constructs stabilization with non-enzyme labelled antibody combination immune magnetic Nano microsphere Property the strong, electrochemical immunosensor that has a wide range of application, reach quickly detection Listeria Monocytogenes.
The detection method of the present invention using redox graphene be the fixed AuNPs of substrate as electrochemical label, in order to avoid Epidemic disease magnetic Nano microsphere (Immunomagnetic nanoparticles, IMNPS) is capture probe, for being enriched with measuring samples In object.Redox graphene/nanogold (rGO/AuNPs) to modify Anti-L.monocytogenes antibody is made Interlayer structure is formed for non-enzyme marker and the IMNPS of enrichment L.monocytogenes.It will be sandwich under the action of externally-applied magnetic field Structural composites are adsorbed on the surface screen printing carbon electrode (Screen Printed Carbon Electrode, SPCE), with difference Pulse voltammetry (Differential Pulse Voltammetry, DPV) is divided to be analyzed.DPV response signals are in 103CFU/ ML-109CFU/mL has good linear relationship, equation of linear regression y=0.02933x-0.01996, R2=0.99367, inspection Rising limit is 1.8 × 104CFUmL-1(S/N=3), i.e., 54CFU in the detection sample of 3 μ L in the detection L.monocytogenes can be detected.Electrochemical immunosensor prepared by the present invention, it is at low cost, it is easy to operate, in reality There is good foreground in the application of border.
Description of the drawings
Fig. 1 is the agglutination characterization schematic diagram of IMNPS.
Fig. 2 is the ultraviolet-visible spectrogram of GO, rGO and rGO/AuNPs.
Fig. 3 is the DPV curve graphs in the different modifying stage on immunoelectrode.(A：Bare electrode, B：Modification L.monocytogenes/IMNPS, C：Modify rGO/AuNPs/Ab/L.monocytogenes/IMNPS.Test bottom liquid is deoxygenation 0.2M HCl).
Fig. 4 be IMNPS from L.monocytogenes the corresponding reduction peak current value of different incubation times curved line relation Figure.
Fig. 5 is rGO/AuNPs/Ab electric in the corresponding reduction peak of different incubation times from L.monocytogenes/IMNPS The curve relation figure of flow valuve.
Fig. 6 is the correspondence figure of immunoelectrode reduction peak current value and L.monocytogenes log concentration values.
Fig. 7 is the canonical plotting of immunoelectrode reduction peak current value and L.monocytogenes log concentration values.
Fig. 8 is the specific schematic diagram of immunoelectrode.
Specific implementation mode
Embodiment 1
One, Fe3O4/SiO2–NH2The preparation of nanoparticle
Fe is prepared using coprecipitation method3O4Magnetic Nano material.Synthetic method is as follows：2.70mM FeCl3It is dissolved in In 70mL distilled water, 1.35mM FeSO are then added4, it is eventually adding NH3·6H2O magnetic in the case where oxygen free condition ensures 80 DEG C simultaneously Power stirs 60min.It is washed respectively 3 times using ethyl alcohol and distilled water, 60mL is settled to after the completion of washing.Successfully prepare 60mLFe3O4Magnetic Nano material.
60mL absolute ethyl alcohols, 10mL distilled water, 1mL TEOS and 9mL NH are added in the reaction vessel3·6H2O, stirring are equal The above-mentioned preparation Fe of 1mL are added after even3O4Magnetic Nano material stirs at 25 DEG C for 24 hours, and washing is repeatedly settled to 1mL and can be obtained Fe3O4/SiO2Magnetic Nano material.40mL absolute ethyl alcohols, 160 μ L APTES and 1mL Fe are added in the reaction vessel3O4/ SiO2It stirs at 25 DEG C of magnetic Nano material for 24 hours, is washed respectively 3 times using ethyl alcohol and PBS, the straight 1mL of constant volume, you can obtain Fe3O4/SiO2–NH2Magnetic Nano material.
Two, Ab/SiO2/Fe3O4Preparation
Take 250 μ L Fe3O4/SiO2–NH22.5% glutaraldehyde is added in magnetic Nano material, and (25 DEG C) are protected from light magnetic at room temperature Power stirs 3h, and PBS is washed 3 times after amino-reactive, and is resuspended in PBS.It is added 50 μ L Anti-L.Monocytogenes (Ab), 25 2h is stirred at DEG C, PBS is cleaned 3 times.Then the 0.2%BSA of 1mL is taken, 1.5h is stirred at room temperature and carries out nonspecific binding site Closing is resuspended in 4mL PBS to get IMNPS (Ab/SiO after cleaning 3 times2/Fe3O4), it is in store at 4 DEG C.
To verify SiO2/Fe3O4Whether modified respectively with Anti-L.monocytogenes successfully, carries out agglutination and test Card.Process is as follows：10 μ L IMNPS are taken to be added in left and right agglutination slide respectively, 10 μ L PBS are added in left side, and purpose is added in right side Bacterium L.monocytogenes (108CFU·mL-1), 20s is quickly stirred, agglutination phenomenon is observed, phenomenon is as shown in Figure 1.By being aggregated As a result known to：Compared with PBS blank controls, there is apparent agglutination patch in right side, and solution becomes clarification, Anti- L.monocytogenes successfully modifies SiO2/Fe3O4On.
Three, the preparation of rGO/AuNPs/Ab
Using one-step synthesis method rGO/AuNPs nano materials.4mg graphene oxides are taken to be dissolved in 100mL distilled water, ultrasound Decentralized processing 3h.It takes HAuCl4 (2.5mM) to be added in mixed liquor, is heated to boiling, be rapidly added one timing of sodium citrate reaction Between.Centrifuge washing is colourless to supernatant, is resuspended in PBS, is in store at 4 DEG C to get to rGO/AuNP nano materials.Using Ultraviolet specrophotometer measures GO, rGO and rGO/AuNPs respectively, to characterize the synthesis of rGO/AuNPs compounds.As a result such as Shown in Fig. 2, within the scope of the absorbing wavelength of 200-800nm, GO has apparent absorption due to the transition of the π-π * of C=C at 230nm Peak (curve b)；GO restores to obtain rGO, and the absorption peak red shift of rGO restores to 245nm to the pi-conjugated structures of π-(curve a).It is making After standby rGO/AuNPs compounds, it is the absorption peak of rGO that compound has absorption peak, 248nm at 248nm and 518nm, and 518nm is Absorption Characteristics peak (the curve c) of apparent AuNPs.Illustrate that AuNPs is successfully attached to the surfaces rGO, forms rGO/AuNPs compounds.
It takes 4mL rGO/AuNPs nano materials that 40 μ L Anti-L.monocytogenes (Ab) are added, is incubated at 4 DEG C 12h is then centrifuged for 3 times, and the 0.2%BSA that 4mL is added is incubated 1h progress nonspecific binding site closings under the conditions of 4 DEG C, from The heart washs 3 times, is resuspended in 2mL PBS, is in store at 4 DEG C to get to rGO/AuNPs/Ab nano materials.
Four, the preparation of electrochemical immunosensor and testing principle
Immunosensor is prepared using double-antibody method：Object can be captured using biomolecular from complicated sample To reach enrichment purpose, 40 μ L IMNPS are added in 1mL sample solutions, are incubated 40min at 25 DEG C after mixing, PBS is washed It washs 3 times.The compound of IMNPS immuno absorbences L.monocytogenes is obtained, 40 μ L rGO/AuNPs/Ab are added, mixing is equal 40min is incubated after even at 25 DEG C, PBS is washed 3 times, is resuspended in 40 μ L PBS to get to rGO/AuNPs/Ab/ L.monocytogenes/IMNPS.3 μ L rGO/AuNPs/Ab/L.monocytogenes/IMNPS are taken to be added drop-wise to silk-screen printing Electrode working electrode surface, while magnet is placed at the working electrode back side, ensure rGO/AuNPs/Ab/L.monocytogenes/ IMNPS compounds are firmly adsorbed on working electrode surface, i.e., successfully prepare immunosensor using double-antibody method.In electrode Surface is added dropwise 100 μ L 0.2M HCl and tests bottom liquid, carries out DPV detections.
Detection method and principle：Using CHI 660C electrochemical workstations for working electrode surface (wherein work in SPCE It is immunoelectrode to make electrode, is carbon electrode to electrode, and reference electrode is Ag/AgCl electrodes), it carries out characterization with DPV and qualitative sentences Disconnected immunoelectrode.
The setting of DPV detection methods：Oxidizing potential 1.25V, oxidization time 120s, DPV electric potential scanning ranging from 1.0V- 0.0V, testing principle：Under oxidizing potential, in the rGO/AuNPs/Ab/L.monocytogenes/IMNPS of working electrode surface Au is tested in deoxygenation in the liquid 0.2M HCl of bottom, is oxidized to Au3+.The Au under the effect of DPV electrochemical methods3+It is reduced to Au, is generated The process of electron transmission realizes testing goal.
Immunosensor is occurred with rGO/AuNPs/Ab/L.monocytogenes/IMNPS on the working electrode of SPCE Redox reaction, to generate corresponding current signal.The reason of signal is generated in principle is added dropwise in working electrode surface What the AuNPs in compound was generated, in order to exclude the influence of other nano materials in background and compound, carry out electrode surface not With the DPV characterizations of modification, carries out in optimal conditions, 100 μ L 0.2M HCl are added dropwise respectively in A:Bare electrode, B:Modification L.monocytogenes/IMNPS、C：Modify rGO/AuNPs/Ab/L.monocytogenes/IMNPS.The results are shown in Figure 3.
In the case where modifying condition C, occurs apparent reduction peak at 0.34V；At condition A and condition B, all without reduction at 0.34V Peak, it was demonstrated that response signal derives from non-enzyme marker AuNPs in the immunosensor prepared in the present invention, and is not repaiied by other The influence of material and test bottom liquid during decorations.
Embodiment 2
1, the optimization of IMNPS and Listeria Monocytogenes incubation time
IMNPS and L.monocytogenes is in suitable incubation time, the magnetic Nano material and purpose of antibody modification Bacterium specifically binds more secured.As shown in Figure 4：With the variation of IMNPS and L.monocytogenes incubation times, exempt from The peak point current of the reduction peak of epidemic disease electrode also has corresponding variation.With when incubation time increases in 10min to 40min The peak point current of reduction peak gradually increases, wherein incubation time be 30min when, with incubation time be 20min when peak current Value has smaller reduction, it may be possible to during antibody and purpose bacterium are specifically bound, in incubation time after 20min, The antibody location that magnetic ball surface is not specifically bound is reduced, during antigen is combined with antibody, antigenic competition magnetic ball surface Antibody location is that peak point current slightly reduces, but continuing growing with incubation time, antigen are combined increase with antibody, Peak point current is maximum when 40min, is then gradually reduced again.So selecting to be incubated 40min to be IMNPS and L.monocytogenes Incubation time.
2、rGO/AuNPs/Ab2With the optimization of IMNPS/L incubation times
Ibid, the antibody of the compound substance markers of rGO/AuNPs can be enriched in suitable incubation time with IMNPS L.monocytogenes is specifically bound to greatest extent, to obtain larger response signal.As shown in Figure 5：Incubation time exists Peak point current is maximum under the conditions of 40min, so selecting to be incubated 40min to be rGO/AuNPs/Ab and L.monocytogenes/ IMNPS incubation times.
3, response signal graph of the immunosensor to the Listeria Monocytogenes of various concentration gradient
Use the detection method immunoelectrode of DPV to the monocyte hyperplasia Li Si of various concentration gradient in optimal conditions Special Salmonella detects to obtain corresponding peak point current, as shown in fig. 6, with the increase of bacteria concentration, antigen with modify anti-on IMNPS Body specific binding increases, corresponding to go back to specifically bind the amount increase to form interlayer structure with rGO/AuNPs/Ab Parent peak peak point current also gradually increases.Meanwhile can be obtained by Fig. 7, it is 10 in bacteria concentration3CFU/mL-109CFU/mL is peak point current Logarithm with bacteria concentration is at good linear relationship, linear relationship y=0.02933x-0.01996, R2=0.99367, inspection Rising limit is 1.8 × 104CFU·mL-1(S/N=3), i.e., 54CFU L.monocytogenes in the detection sample of 3 μ L in the detection It can be detected.
4, specific test
Its specificity determines anti-interference in practical applications in the research of immunosensor, selects in the present invention S.aureus, B.subtilis, C.freundii are selected, S.sonnei is interference bacterium, using PBS as blank control, carries out specificity Detection, the results are shown in Figure 8.Using peak point current size as criterion, the Ipc=of purpose bacterium L.monocytogenes The Ipc=0.024 of Ipc=0.062 the μ A, B.subtilis of 0.221 μ A, PBS blank control Ipc=0.042 μ A, S.aureus The peak point current of Ipc=0.04 the μ A, L.monocytogenes of Ipc=0.058 the μ A, S.sonnei of μ A, C.freundii are bright It is aobvious to be more than PBS blank controls and other four kinds of bacterium, it was demonstrated that the immunosensor has specificity.
3 sample detection of embodiment
Accuracy for the immunosensor prepared in the verification present invention, object is detected by actual sample of milk, according to The method of the present invention is detected, and rate is recycled according to standard curve, to judge its accuracy.In milk be added 2.9 × 107The L.monocytogenes of CFU/mL concentration, it is 78% to measure the rate of recovery according to foregoing invention method.There is rate of recovery result Display the present invention in design method can preferably detect the L.monocytogenes in skim milk, be one kind have compared with The immunosensor of good advantage.
1 actual sample of table detects
In order to further verify the practicability of the immunosensor, using Blind Test method；Detailed process is as follows：To each reality Sample is numbered, and is divided to two groups to be detected, and wherein A groups detect actual sample using the immunosensor prepared, and B groups use state Mark (GB4789.30-2010) is detected, and number person does not participate in two groups of detections of A, B, and forbids mutually exchanging.Experimental result is such as Shown in table 2, the immunosensor of preparation has the accuracy rate of 93% (n=30), illustrates to have good accuracy and actually answer With value.
2 immunoelectrode Accuracy Verification result of table
The present invention is restored GO and HAuCl4 using sodium citrate as one step of reducing agent and prepares rGO/AuNPs, in rGO/AuNPs Antibody is modified as with the non-enzyme labelled antibody of AuNPs signal designation objects, L.monocytogenes is specifically bound, avoids enzyme Mark the influence of the destabilizing factors to immunosensor stability such as enzyme easy in inactivation in electrochemical sensor.It is rich using IMNPS simultaneously Collection separation L.monocytogenes, keeps preparing for immunosensor more convenient, and sensitivity is also improved.It is detecting Middle IMNPS/L.monocytogenes/rGO/AuNPs is added dropwise the SPCE under with magnetic fields and carries out, and avoids electrode table The cumbersome modification in face, it is more quick and convenient.
Be detected with optimal conditions, immunosensor prepared by the present invention have apparent signal amplification and preferably Accuracy；The range of linearity for detecting L.monocytogenes is 103CFU/mL-109CFU/mL, detection is limited to 1.8 × 104CFU·mL-1(S/N=3), i.e., 54CFU L.monocytogenes can be detected in the detection sample of 3 μ L in the detection. Non- purpose bacterium current peak is being detected significantly lower than purpose by the sensor prepared in the present invention known to specific detection simultaneously Bacterium.So the immunosensor of the quick detection L.monocytogenes prepared in the present invention has preferable accuracy, sensitive Degree and reproducibility.It can be used for the quick screening of microorganism, clinical diagnosis and food security etc..

Claims (1)

1. a kind of method of the quick detection Listeria Monocytogenes of non-diagnostic purpose, which is characterized in that including with Lower step：
（1）Prepare Fe3O4/SiO2–NH2Nanoparticle：FeCl3It is dissolved in distilled water, FeSO is added4, it is eventually adding NH3· 6H2O, 60 min of magnetic agitation, obtains Fe under anaerobic3O4 Magnetic Nano material；Anhydrous second is added in the reaction vessel Alcohol, distilled water, TEOS and NH3· 6H2O is stirring evenly and then adding into Fe3O4 Magnetic Nano material stirs 24 h at 25 DEG C, obtains Fe3O4/SiO2Magnetic Nano material；Absolute ethyl alcohol, APTES and Fe are added in the reaction vessel3O4/SiO2Magnetic Nano material Material；24 h are stirred at 25 DEG C, obtain Fe3O4/SiO2–NH2Nanoparticle；
（2）Prepare Ab/SiO2/Fe3O4：Fe3O4/SiO2–NH22.5% glutaraldehyde is added in nanoparticle, and 25 DEG C are protected from light magnetic force and stir 3 h are mixed, Anti- is added after amino-reactiveL. Monocytogenes(Ab), it stirs at 25 DEG C and closes non-spy with BSA after 2 h Anisotropic binding site, obtains Ab/SiO2/Fe3O4(IMNPS)；
（3）Prepare rGO/AuNPs/Ab：Graphene oxide is dissolved in distilled water, and HAuCl4 is added, is heated to boiling, rapid to add Enter sodium citrate reaction, obtains rGO/AuNP nano materials；Anti- is added into rGO/AuNPs nano materialsL. monocytogenes (Ab), it is incubated 12 h at 4 DEG C, closes nonspecific binding site with BSA, obtains rGO/AuNPs/Ab and receive Rice material；
（4）Build double-antibody method：Step is added in testing sample solution（2）Gained Ab/SiO2/Fe3O4(IMNPS), it mixes It is incubated 40min at 25 DEG C after uniformly, PBS is washed, and obtains IMNPS immuno absorbencesL. monocytogenesCompound, add Step（3）Gained rGO/AuNPs/Ab, 40min is incubated at 25 DEG C after mixing, and PBS washings obtain rGO/AuNPs/Ab/L. monocytogenes/ IMNPS；Take rGO/AuNPs/Ab/L. monocytogenes/ IMNPS is added drop-wise to screen printing electrode Working electrode surface, while magnet is placed at the working electrode back side, ensure rGO/AuNPs/Ab/L. monocytogenes/ IMNPS compounds are firmly adsorbed on working electrode surface；
（5）DPV is detected：100 μ L 0.2M HCl are added dropwise in working electrode surface and test bottom liquid, DPV detections are carried out, wherein to electricity Extremely carbon electrode, reference electrode are Ag/AgCl electrodes, are characterized with DPV and qualitatively judge immunoelectrode；The detection sides DPV The setting of method：1.25 V of oxidizing potential, 120 s of oxidization time, DPV electric potential scannings ranging from 1.0V-0.0V；Obtain peak current Value y；
（6）As a result judge：Peak point current y is substituted into formula：Y=0.02933x-0.01996 is calculatedL. Monocytogenes Bacteria concentration x values.
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